Influence of divalent cations on inner-arm mutants of restriction endonuclease EcoRI

To understand the functional role of the inner arm region of EcoRI for the interaction with its DNA substrate, we mutated each of the positively charged amino acid residues Lys130 and Arg131 to Ala or Glu. The resulting cleavage activities are only about tenfold reduced but DNA binding in the absence of divalent cations is totally disrupted for three of the mutants ([Ala130]EcoRI, [Glu130]EcoRI and [Glu131]EcoRI). The binding can be rescued by adding 1 mM Ca2+. This binding behaviour resembles that of the blunt end cutter EcoRV. Furthermore, the cleavage activity of the [Glu130]EcoRI is stimulated by Ca2+. Therefore, a second metal binding site is involved in [Glu130]EcoRI catalyzed DNA cleavage. Its location is postulated to be in the inner arm region at positions 133 and 135 presenting an Asp-Xaa-Asp motif typical for Ca2+ binding. A net positive charge at the tip of the inner arm due to the basic amino acids Lys130 and Arg131 in the wild-type enzyme or due to binding of a divalent cation in the mutants is important for DNA recognition by EcoRI. However, a direct phosphate contact providing an indirect readout is not observed. Implications of the binding and cleavage behaviour with regard to mechanistic differences between blunt end and sticky end cutters and a general catalytic mechanism of restriction enzymes are discussed.